title: Novel and sensitive ELISA for the rapid quantification of recombinant p64K protein.
abstract: The antigenic P64k protein from the pathogenic bacterium Neisseria meningitidis has been used as an immunological carrier in several conjugated vaccines. The aim of this report was to develop and validate a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of recombinant p64k protein, to perform both manufacturing process and identification in different vaccine preparations. Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH). The reference curve showed to be precise and accurate over the entire linear range of 1.25 and 20ng/mL with a limit of quantification validated to 1.25ng/mL. The intra- and inter-assay coefficient of variation ranged from 0.35 to 6.65% and 4.70 to 10.63%, respectively. The ANOVA test used in the specificity/interference study revealed parallelism among curves (p>0.1), which indicates the lack of interference in the working range. Recovery obtained from the accuracy test, using three concentration levels, varied between 94 and 111%, confirming the assay's reliability. The short-term study shown the P64k is stable to -20°C up to 1-week. This ELISA was fully used to assess its manufacturing process and molecular interaction issues in several vaccine preparations. Thus, this immunoassay could be an excellent analytical choice to characterize the quality of that recombinant protein in several contexts as manufacturing process and molecular conjugates.
authors.affiliation: Process Control Department, Center for Genetic Engineering and Biotechnology, Ave 31 between 158 and 190, P.O. Box 6162, Havana 10600, Cuba. alberto.leyva@cigb.edu.cu
authors.forename: Alberto
authors.initials: A
authors.lastname: Leyva
authors.forename: Julio C
authors.initials: JC
authors.lastname: Sánchez
authors.forename: Denis
authors.initials: D
authors.lastname: Alvarez
authors.forename: Bárbara
authors.initials: B
authors.lastname: Pérez
authors.forename: Lissette
authors.initials: L
authors.lastname: López
authors.forename: Shaily
authors.initials: S
authors.lastname: Paz
authors.forename: Edel
authors.initials: E
authors.lastname: Torres
authors.forename: Tatiana
authors.initials: T
authors.lastname: González
authors.forename: Milagros
authors.initials: M
authors.lastname: Font
authors.forename: Neyda
authors.initials: N
authors.lastname: Hernández
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Valdés
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000235:genetics; D001428:Bacterial Vaccines; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D057230:Limit of Detection; D009345:Neisseria meningitidis / Q000276:immunology*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; C112623:lpdA protein, Neisseria meningitidis